WO2011152098A1 - Developing solution for immunochromatography method, and measurement method using same - Google Patents

Abstract

Disclosed is a means which enables the achievement of an accurate and highly sensitive immunochromatography method that can detect an analyte satisfactorily even when the analyte is contained at a low concentration. Specifically disclosed is a developing solution for use in an immunochromatography method using a detection reagent that is labeled with an insoluble carrier, which is characterized by containing a nucleic acid molecule. A preferred example of the insoluble carrier is a silica particle. The silica particle preferably has a fluorescent compound chemically bound thereto or adsorbed thereon.

Description

Developing solution for immunochromatography and measurement method using it

The present invention relates to an immunochromatographic method which is one of immunoassay methods capable of detecting a non-detected substance contained in a biological sample with high sensitivity, and particularly to a developing solution constituting a moving bed in the method.

In the immunochromatography method, a substance (for example, a first antibody against the antigen) that specifically binds to a substance to be detected (for example, an antigen) contained in a sample such as blood, saliva, or urine and the substance are combined. A conjugate consisting of a labeling substance to be labeled is reacted, a mobile phase containing the formed complex is developed into a stationary phase by the principle of chromatography, and an immobilized reagent (for example, against the antigen) is reacted at a reaction site on the stationary phase. This is a technique for qualitatively or quantitatively analyzing a substance to be detected in the sample, wherein a signal emitted from the labeling substance is detected after the conjugate is supplemented with a second antibody).

As a labeling substance in such an immunochromatography method, for example, colloidal metal particles (for example, gold particles), colored latex particles and the like are conventionally used. These labeling substances have the advantage of being able to make positive / negative judgments visually, but further enhancement of sensitivity is promising for the expansion of the application of immunochromatographic methods. It is considered that a method of detecting fluorescence with a detector using the above is effective. Metal particles are strongly colored, but in principle do not emit fluorescence. Further, as latex particles, there are fluorescent latex particles into which a fluorescent dye is introduced. However, it is difficult for antibodies to bind to the particle surface (that is, it is difficult to form a conjugate), and nonspecific binding due to hydrophobic bonds is likely to occur. Therefore, in recent years, silica particles having high hydrophilicity and high antibody binding properties in which fluorescent compounds such as semiconductor nanoparticles and fluorescent dyes are encapsulated are expected as new labeling substances. .

Now, when insoluble carriers such as silica particles, conventional colloidal metal particles, and colored latex particles are used as a labeling substance, (1) the detection reagent (particularly the insoluble carrier portion) does not aggregate, Surely develop and move on the chromatographic medium to reach the reaction site, and (2) suppress false positive reactions caused by non-specific adsorption of components other than the substance to be detected contained in the sample, Etc. are required.

As means for satisfying the above requirements, for example, in Patent Document 1 (Japanese Patent Laid-Open No. 2010-019786), a vinyl-based water-soluble polymer having an oxygen atom and a nitrogen atom-containing polar group and an HLB value of 13 to 18 are used. A developing solution in an immunochromatographic method using a detection reagent labeled with an insoluble carrier containing a nonionic surfactant is described. Patent Document 2 (Japanese Patent Laid-Open No. 2009-162537) discloses a silica in which a polysaccharide is adsorbed on the surface of silica particles and a biomolecule is adsorbed or covalently bonded to the surface of the silica particles and the outside of the polysaccharide. There are described particle / polysaccharide / biomolecule composite particles and analytical reagents using composite particle colloids in which such composite particles are dispersed in a dispersion medium.

JP 2010-019786 A JP 2009-162537 A

The present invention provides accurate and high sensitivity capable of sufficiently detecting even a low-concentration detected substance by suppressing a decrease in measurement sensitivity due to aggregation of insoluble carriers and nonspecific adsorption. It is an object of the present invention to provide a means for enabling an immunochromatographic method.

The present inventors have found that the above-mentioned problems can be solved by using a solution containing a nucleic acid molecule as a developing solution in an immunochromatographic method using a detection reagent labeled with an insoluble carrier, and the present invention has been completed. I came to let you.

That is, in one aspect, the present invention provides a developing solution in an immunochromatographic method using a detection reagent labeled with an insoluble carrier, characterized by containing a nucleic acid molecule. *

For example, silica particles are suitable as the insoluble carrier. The silica particles are preferably those in which a fluorescent compound is chemically bonded or adsorbed. On the other hand, the nucleic acid molecule is preferably composed of, for example, one or more nucleotides.

In another aspect, the present invention provides an immunochromatographic method characterized in that the developing solution is used as a developing solution constituting a moving bed or as a sample diluent.

In a further aspect of the present invention, there is provided an immunochromatographic detection kit comprising at least the above developing solution and a chromatographic medium.

By using the developing solution of the present invention containing a nucleic acid molecule, an accurate and highly sensitive immunochromatographic method capable of sufficiently detecting even a low-concentration target substance becomes possible. The following inference does not limit the present invention, but the nucleic acid molecule added to the developing solution has a function of masking an insoluble carrier (for example, using silica particles), thereby insoluble. It is presumed that the decrease in measurement sensitivity due to carrier aggregation and nonspecific adsorption is suppressed.

As one aspect of the present invention, a “nucleic acid molecule” is added in advance to a developing solution constituting a moving layer on a chromatographic medium, while a developing phase moving path of a mobile phase in a chromatographic medium constituting a stationary phase. In other words, the "detection reagent labeled with an insoluble carrier" is present in the region between the end portion to which the mobile phase of the chromatographic medium is applied and the reaction site, and the developing solution is allowed to flow on the chromatographic medium. One example is that the “detection reagent labeled with an insoluble carrier” dissolves when developed.

The above aspects include the following two aspects, for example.

In one embodiment (hereinafter sometimes referred to as “first embodiment of the present invention”), a “developing solution addition site” for adding a developing solution and a sample containing a substance to be detected are added to the chromatographic medium. A “sample addition site” for addition, a “detection reagent holding site” where a detection reagent labeled with an insoluble carrier is supported, and a reaction site are arranged in this order. Then, a developing solution containing nucleic acid molecules is added to the developing solution addition site and developed on the chromatographic medium.

In the other aspect (hereinafter sometimes referred to as “second aspect of the present invention”), the above-mentioned “developing liquid addition site” is not disposed on the chromatographic medium, and “sample addition site” The “detection reagent holding site” and the “reaction site” are arranged in this order. Then, the developing solution of the present invention containing nucleic acid molecules is used as a “sample diluent” for diluting the sample, and the sample thus diluted (mixed with the developing solution of the present invention) is added to the “sample addition” It is added to "site" and developed on a chromatographic medium.

As another aspect of the present invention (hereinafter, sometimes referred to as “third aspect of the present invention”), the mobile phase in the chromatographic medium constituting the stationary phase, ie, the mobile phase of the chromatographic medium. `` Nucleic acid molecule '' and `` detection reagent labeled with an insoluble carrier '' are present in the region between the end to which the reagent is applied and the reaction site. Is dissolved in the developing solution of the present invention containing “nucleic acid molecule” together with “detection reagent labeled with an insoluble carrier”.

In addition to these exemplified embodiments, if the nucleic acid molecules in the developing solution can come into contact with the detection reagent, particularly the site of the insoluble carrier in the stage of development on the chromatographic medium, such a developing solution can be used. And measuring methods are included in the present invention.

Developing solution / Nucleic acid molecule The developing solution in the immunochromatography method is a liquid that constitutes a mobile phase, and moves on a chromatographic medium that is a stationary phase together with a sample containing a substance to be detected and a detection reagent labeled with an insoluble carrier To do. The developing solution of the present invention is mainly characterized by containing a nucleic acid molecule, and other constituents of the developing solution, for example, components in the developing solution such as a detection reagent labeled with an insoluble carrier, and the developing solution. The method of using the liquid can be the same as that in the conventional immunochromatographic method as described in, for example, the above-mentioned Patent Document 1 (Japanese Patent Laid-Open No. 2010-19786).

Further, as shown in the second aspect of the present invention described above, the developing solution of the present invention can also be used as a sample diluent, and the sample diluted as such is directly developed on an immunochromatographic medium. Can be made. The description regarding the “developing solution” in the present specification is applicable to the case of using as a “sample diluent” unless otherwise specified.

As the “nucleic acid molecule” contained in the developing solution of the present invention, a nucleoside, a nucleotide, and an oligomer or polymer in which two or more nucleotides are bonded can be used. The nucleoside or nucleotide includes both ribonucleoside or ribonucleotide and deoxynucleoside or deoxyribonucleotide, and the number of phosphate groups in the nucleotide may be any of 1, 2 or 3. . As such a nucleic acid molecule, one consisting of at least one kind of nucleotide, for example, dNTP-Mixture generally commercially available and used as a substrate for DNA polymerase in a DNA synthesis reaction is suitable. In an immunochromatographic method using a nucleic acid as a substance to be detected using a probe (DNA or RNA having a complementary base sequence) immobilized on a chromatographic medium, such a false positive reaction is avoided. The addition of oligomers or polymers that may be adsorbed to the probe as nucleic acid molecules to the developing solution of the present invention should be avoided.

The concentration of the nucleic acid molecule in the developing solution can be appropriately adjusted according to the mode of the nucleic acid molecule and the insoluble carrier, and is not uniformly defined. For example, the dNTP Mixture is used as the nucleic acid molecule, and the insoluble carrier is used. When silica particles are used, it is preferable to use 0.1 to 10% by weight of nucleic acid molecules with respect to the silica particles.

Moreover, it is preferable that the developing solution of the present invention usually contains water as a solvent and contains a buffer in addition to the nucleic acid molecule. As the buffer, for example, phosphate, trishydroxymethylaminomethane, Good's buffer and the like are preferable. With such a buffer, the pH of the developing solution of the present invention is preferably adjusted in the range of 6 to 8.5. Furthermore, as other components, protein components such as bovine serum albumin (BSA) (content is usually 0.01 wt% to 10 wt%) may optionally be included.

Chromatographic medium The chromatographic medium used in the immunochromatographic method of the present invention is an inert reagent composed of a microporous material exhibiting capillary action, as in the conventional general chromatographic medium, and is used as a detection reagent. The material is not particularly limited as long as it does not react with the immobilization reagent, the substance to be detected, and the like, and has a developing speed at which sufficient sensitivity can be obtained by a determination in a short time.

Examples of the chromatographic medium include silica, titania, zirconia, ceria, alumina and other ceramic fine particles or organic polymer fine particles, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, nitrocellulose, cellulose acetate, etc. Examples thereof include a fibrous or non-woven fibrous matrix composed of a cellulose derivative and the like, a membrane, a filter paper, a glass fiber filter paper, a cloth, and cotton. Even if the microparticles are not porous per se, voids are generated between the microparticles in the packed state and function as a chromatographic medium. Cellulose derivatives and nylon membranes, filter papers, glass fiber filter papers and the like are preferred, and nitrocellulose membranes, mixed nitrocellulose ester (mixtures of nitrocellulose and cellulose acetate) membranes, nylon membranes, and filter papers are more preferred.

The form and size of the chromatographic medium used for carrying out the present invention are not particularly limited, and may be appropriate in terms of actual operation and observation of reaction results. In order to make the operation easier, it is preferable to provide a support made of plastic or the like on the back surface of the chromatographic medium having the reaction sites formed on the surface. The properties of the support are not particularly limited, but when the measurement result is observed by visual judgment, the support preferably has a color that is not similar to the color caused by the labeling substance. Usually, it is colorless or white.

In addition to the chromatographic medium, if necessary, a sample addition site (sample pad, etc.) for adding a sample containing the substance to be detected, a site for removing solid components such as blood cells in the sample (blood cell separation site, etc.) ), A developing solution addition site for adding a developing solution, an absorption site (absorption pad, etc.) that absorbs a detected substance or developing solution that has not been captured in the reaction site, a control site that indicates that the measurement has been performed normally, etc. May be incorporated. The members of these parts are not particularly limited as long as the sample solution and the developing solution can move by capillary action. Generally, the members are made of a plurality of porous materials such as a nitrocellulose membrane, filter paper, and glass fiber filter paper. A material suitable for the purpose can be selected and used, and can be arranged so as to be connected to the chromatographic medium on which the immobilization reagent is immobilized by a capillary.

A detection kit for the immunochromatography method can also be constituted by a combination of such a chromatographic medium and a developing solution as described above. In this case, for example, as shown in the first to third aspects of the present invention described above, the chromatographic medium is formed with a predetermined portion corresponding to a developing solution used in combination or a measuring method used. do it. In addition, the detection kit may further include other components and instructions showing the usage method.

Immobilization reagent / reaction site A reaction site in which a substance that specifically binds to the substance to be detected, for example, an antibody is immobilized as an immobilization reagent at an arbitrary position is formed on the chromatographic medium used in the present invention. The immobilization reagent can be immobilized on the chromatographic medium by directly immobilizing the immobilization reagent on the chromatographic medium by physical or chemical means, and by physically or chemically immobilizing the immobilization reagent on fine particles such as latex particles. There is an indirect immobilization method in which these fine particles are chemically bound and captured and immobilized on a chromatographic medium.

As a direct immobilization method, physical adsorption may be used, or covalent bonding may be used. In general, physical adsorption can be utilized when the chromatographic medium is a nitrocellulose membrane or a mixed nitrocellulose ester membrane. In addition, in the covalent bond, cyanogen bromide, glutaraldehyde, carbodiimide and the like are generally used for activating the chromatographic medium, but any method can be used.

On the other hand, as an indirect immobilization method, there is a method in which an immobilization reagent is bound to insoluble fine particles and then immobilization on a chromatographic medium. The particle size of the insoluble fine particles can be selected such that it is captured by the chromatographic medium but cannot move, and is preferably fine particles having an average particle size of about 10 μm or less. Various particles used for antigen-antibody reaction are known as these particles, and these known particles can also be used in the present invention. For example, organic polymers such as organic polymer latex particles obtained by emulsion polymerization methods such as polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer. Examples include fine particles of substances, fine particles such as gelatin, bentonite, agarose, and crosslinked dextran, inorganic oxides such as silica, silica-alumina, and alumina, and inorganic particles obtained by introducing functional groups into inorganic oxides by silane coupling treatment, and the like. . In the present invention, direct immobilization is preferred from the standpoint of ease of sensitivity adjustment.

In addition, various methods can be used to immobilize the immobilization reagent on the chromatographic medium. For example, various techniques such as a microsyringe, a pen with a control pump, and ink jet printing can be used. The form of the reaction site is not particularly limited. For example, in the case of an immunochromatographic medium of the type that visually confirms the presence of a substance to be detected, a circular spot, a line extending in a direction perpendicular to the chromatographic medium, numbers, letters, It can also be fixed as a symbol such as + or-.

After the immobilization reagent is immobilized, the chromatographic medium can be subjected to a blocking treatment by a known method as necessary in order to prevent the accuracy of the analysis from being reduced due to nonspecific adsorption. Generally, proteins such as bovine serum albumin, skim milk, casein, and gelatin are preferably used for the blocking treatment. After such blocking treatment, a surfactant such as Tween 20, Triton X-100, or SDS may be washed in combination with one or more if necessary.

Detection reagent / detection reagent holding site labeled with an insoluble carrier The detection reagent used in the present invention is a substance that specifically binds to a substance to be detected, such as an antibody, and insoluble as a labeling substance for labeling it. It is a conjugate with a carrier.

The insoluble carrier as the labeling substance used in the present invention includes silica particles; colloidal metal particles such as gold, silver and platinum; colloidal metal oxide particles such as iron oxide; colloidal nonmetal particles such as sulfur. Known materials such as latex particles made of a synthetic polymer can be used. Among these, silica particles, particularly silica particles to which fluorescent compounds such as semiconductor nanoparticles and fluorescent dyes are chemically bonded or adsorbed are suitable as the insoluble carrier in the present invention.

As the silica particles as the insoluble carrier of the detection reagent, those prepared by a known method as used in the invention described in the above-mentioned Patent Document 2 (Japanese Patent Laid-Open No. 2009-162537) are used. be able to.

For example, a product obtained by reacting a fluorescent compound with a silane coupling agent, and covalently bonding, ionic bonding, or other chemical bonding or adsorption, is subjected to polycondensation of one or more silane compounds to form a siloxane bond. By forming it, silica particles encapsulating the fluorescent compound in a form in which the fluorescent compound is coated with a layer formed by siloxane bonding between the organosiloxane component and the siloxane component can be produced. More specifically, for example, N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxymethyl group, epoxy group, cyano group, etc. A product obtained by reacting a fluorescent compound having an active group with a silane coupling agent having a substituent (for example, an amino group, a hydroxyl group, or a thiol group) that reacts with the active group, and covalently bonding the product. One or two or more silane compounds may be subjected to condensation polymerization to form a siloxane bond.

Specific examples of the fluorescent compound having the active group include 5- (and -6) -carboxytetramethylrhodamine-NHS ester (trade name, manufactured by emp Biotech GmbH), DY550-NHS ester or DY630-NHS ester ( Examples thereof include a fluorescent dye compound having an NHS ester group such as a trade name, manufactured by Dyomics® GmbH).

Specific examples of the silane coupling agent having a substituent include γ-aminopropyltriethoxysilane (APS), 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane, N-2 Examples thereof include silane coupling agents having an amino group such as (aminoethyl) 3-aminopropylmethyldimethoxysilane and 3-aminopropyltrimethoxysilane. Of these, APS is preferable.

Specific examples of the silane compound to be polycondensed include tetraethoxysilane (TEOS), γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS), 3-thiocyanatopropyltriethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane Can be mentioned. Among these, TEOS is preferable from the viewpoint of forming the siloxane component inside the silica particles, and MPS or APS is preferable from the viewpoint of forming the organosiloxane component inside the silica particles.

In addition, a detection reagent using core-shell type nanoparticles such as Si / SiO ₂ , CdSe / ZnS, CdS / ZnSe as a fluorescent compound can also be produced by a known method. For example, core / shell type nanoparticles of Si / SiO ₂ can be prepared by performing annealing treatment, hydrofluoric acid treatment, and then natural oxidation treatment after sputtering treatment of Si and SiO ₂ . Then, the silane compound as described above and prepared Si / SiO ₂ nanoparticles and (for example TEOS) by condensation polymerization, it is possible to produce silica particles containing the nanoparticles.

By using the method as described above, spherical or nearly spherical silica particles having an appropriate particle size in the immunochromatographic method can be produced. Specifically, the nearly spherical silica particles have a shape in which the ratio of the major axis to the minor axis is 2 or less. The average particle diameter of the silica particles can be adjusted, for example, in the range of 5 nm to 10,000 nm by adjusting the reaction conditions (such as the amount ratio of reactants and the number of reactions). Further, in order to obtain silica particles having a desired average particle diameter, ultrafiltration is performed using an ultrafiltration membrane such as YM-10 or YM-100 (both trade names, manufactured by Millipore), for example, A method of removing particles having a diameter too large or too small, or performing centrifugation at an appropriate gravitational acceleration and collecting only the supernatant or the precipitate may be combined. The average particle diameter of the silica particles can be determined by observing a predetermined number of silica particles using, for example, an electron microscope.

On the other hand, as the colloidal metal, metal oxide or nonmetal particles, and latex particles, commercially available ones or those prepared by a known method may be used. For example, a method for preparing colloidal gold particles includes a method of reducing chloroauric acid with sodium citrate.

A conjugate consisting of a substance that specifically binds to a substance to be detected and an insoluble carrier can be prepared by a known method. Generally, a conjugate is formed by a method of binding them by physicochemical adsorption, a covalent bond, and the like. Can be broadly divided into methods of combining. The former includes, for example, a method in which an antibody is added to a solution in which silica particles or gold particles are dispersed in a colloidal form, and then left for a predetermined time to be physically adsorbed. There is an advantage of being. On the other hand, the latter includes, for example, a method in which a carboxyl group introduced to the particle surface of an insoluble carrier and an amino group of a biomolecule are bonded by an amide bond using a condensing agent, or a so-called cross-linking reagent is used to bind an insoluble carrier and a biomolecule These methods have the advantage that biomolecules can be introduced quantitatively and irreversibly. In addition, after the conjugate is prepared by the method as described above, it is preferable to add a blocking agent such as bovine serum albumin solution to block the particle surface to which the antibody is not bound.

In the actual immunochromatographic method, the detection reagent labeled with an insoluble carrier can be applied dispersed in the developing solution constituting the mobile phase, or the mobile phase in the chromatographic medium constituting the stationary phase can be applied. The present invention can also be applied by causing it to exist on the development movement path, that is, in a region (detection reagent holding site) between the end portion to which the mobile phase of the chromatographic medium is applied and the reaction site. When present on a chromatographic medium, a detection reagent labeled with an insoluble carrier is supported on a predetermined site (detection reagent holding site) so that it can be quickly dissolved in a developing solution and freely moved by capillary action. It is preferable to keep it. In order to improve the resolubility of the insoluble carrier, the detection reagent support is coated with saccharides such as saccharose, maltose, and lactose, and sugar alcohols such as mannitol, or pre-coated with these substances. You can also keep it. When the detection reagent is present on the chromatographic medium by coating / drying, it can be directly coated / dried on the chromatographic medium, or another porous substance such as cellulose filter paper, glass fiber filter paper, nylon, etc. After the detection reagent holding member is formed by applying and drying to a nonwoven fabric, the detection reagent holding member may be connected to the chromatographic medium on which the immobilized reagent is immobilized by a capillary.

Substance to be Detected / Sample In the present invention, the substance to be detected detected by the immunochromatography method is not particularly limited as long as a substance that specifically binds to it is present. Typical substances to be detected are proteins or peptides, but also include nucleic acids, sugars (especially sugar parts of glycoproteins, sugar parts of glycolipids, etc.), complex carbohydrates and the like. In the present invention, “specifically binds” means binding based on the affinity of a biomolecule. Such affinity-based binding includes antigen-antibody binding, sugar-lectin binding, hormone-receptor binding, enzyme-inhibitor binding, complementary nucleic acids and nucleic acid-nucleic acid binding proteins. And the like. Accordingly, when the substance to be detected has antigenicity, the substance that specifically binds to the substance to be detected can be exemplified by a polyclonal antibody or a monoclonal antibody. In addition, when the substance to be detected is a sugar, a lectin protein can be exemplified as a substance that specifically binds to the substance to be detected. Specific detection substances include, for example, carcinoembryonic antigen (CEA), HER2 protein, prostate specific antigen (PSA), CA19-9, α-fetoprotein (AFP), immunosuppressive acidic protein (IPA), CA15- 3, CA125, estrogen receptor, progesterone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), syphilis antibody, Examples include, but are not limited to, influenza virus, human hemoglobin, chlamydia antigen, group A β streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin and the like.

Samples containing the substance to be detected include, for example, biological samples such as whole blood, serum, plasma, urine, saliva, sputum, nasal cavity or throat swab, spinal fluid, amniotic fluid, nipple discharge, tears, sweat, and skin. And exudates from tissues, cells and stool. If necessary, these samples are processed so that a specific binding reaction easily occurs between the substance to be detected and the detection reagent or the immobilization reagent. The treatment method may be either a chemical treatment method using various chemicals such as acids, bases or surfactants, or a physical treatment method using heating / stirring / ultrasonic waves, etc. Also good. For example, when a binding reaction between a substance to be detected and a detection reagent or an immobilization reagent is performed using a region that is not normally exposed on the surface, such as an influenza virus NP antigen, a treatment with a surfactant or the like is performed. Is preferred. As the surfactant used for this purpose, it is preferable to use a nonionic surfactant in consideration of the influence on a specific binding reaction, for example, an antigen-antibody reaction. In addition, these samples may be diluted with a developing solution so that they can be developed in a chromatographic medium as a stationary phase, if necessary.

1. Formation of reaction site on immunochromatographic medium Phosphate buffer solution containing 5% by weight of isopropyl alcohol using 25 × 2.5 cm nitrocellulose membrane (Millipore: HF120) and antibody coater (BioDot) An anti-AFP (alpha-fetoprotein) antibody diluted to a concentration of 1.0 mg / mL at (pH: 7.4) was applied and dried at 50 ° C. for 30 minutes. After drying, the nitrocellulose membrane was immersed in a phosphate buffer solution (pH: 7.4) 200 mL containing 0.5 wt% casein (manufactured by Wako Pure Chemical Industries, Ltd.) at 30 ° C. for 30 minutes for blocking. After blocking, it was washed with a washing solution containing 0.05% by weight of Tween 20 and dried overnight at room temperature to form reaction sites on the chromatographic medium.

2. Preparation of Labeling Substance and Detection Reagent 5- (and -6) -carboxytetramethylrhodamine succimidyl ester (trade name, manufactured by emp Biotech GmbH) 5.8 mg was dissolved in 1 mL of dimethylformamide (DMF). 2.6 μL of APS was added thereto and reacted at room temperature (25 ° C.) for 1 hour. Ethanol 44.8mL, TEOS360mL, distilled water 11.2mL, 28 mass% ammonia water 800μL was added to 150 μL of the obtained reaction solution, and the reaction was performed at room temperature for 24 hours. The reaction solution was centrifuged at a gravitational acceleration of 18000 × g for 30 minutes, and the supernatant was removed. 4 mL of distilled water was added to the precipitated silica particles to disperse the particles, and centrifuged again at a gravity acceleration of 18000 × g for 30 minutes. This washing operation was further repeated twice to remove unreacted TEOS, ammonia and the like contained in the labeled silica nanoparticle dispersion, thereby obtaining rhodamine-containing silica particles.

Next, 0.5 ml of the obtained rhodamine-containing silica particles (particle size 100 nm) is diluted with an anti-AFP (alpha-fetoprotein) antibody diluted to a concentration of 0.1 mg / mL with a phosphate buffer (pH 7.4). 0.1 mL was added and allowed to stand at room temperature for 10 minutes. Subsequently, 0.1 mL of a phosphate buffer solution (pH: 7.4) containing 10% by mass of bovine serum albumin was added and stirred sufficiently, followed by centrifugation at 8000 × g for 15 minutes. After removing the supernatant, 0.1 mL of a phosphate buffer solution (pH 7.4) containing 1% by weight of bovine serum albumin was added to obtain a detection reagent.

3. Preparation of chromatographic medium After the detection reagent prepared as described above was uniformly added to a glass fiber pad, it was dried by a vacuum dryer to obtain a detection reagent holding member. Next, the chromatographic medium in which the reaction sites are formed as described above, the detection reagent holding member, the sample pad used for the portion to which the sample is added, and the developed sample and insoluble carrier are absorbed by the backing sheet. Adhering absorbent pad for. Finally, it was cut with a cutting machine so that the width was 5 mm, and a chromatographic medium was produced.

4). Measurement Using the chromatographic medium prepared as described above, the presence or absence of AFP (alpha-fetoprotein) in the sample was measured by the following method. That is, 0.05% by volume Tween 20, dNTP Mixture as nucleic acid molecule (TaKaRa, Code No. 4030) 25 mmol / L, 1.0% by weight bovine serum albumin and Tris buffer solution (pH: 8.0) containing 150 mM sodium chloride Use the liquid as a negative sample, add a predetermined concentration of AFP (alpha-fetoprotein) to the positive sample, place 100 μL each on the sample pad of the chromatographic medium, and develop the reaction site (after 15 minutes) Signal: S) and fluorescence at non-reactive sites (noise: N) were measured. The results of S / N ratio are shown in Table 1.

5. Comparative Example A dNTP Mixture (manufactured by TaKaRa, Code No. 4030) as a nucleic acid molecule was prepared in the same manner as in the above example except that 25 mmol / l was not blended, and a reaction site was prepared. And fluorescence at non-reactive sites was measured. The results of S / N ratio are shown in Table 1.

Compared to the case of using a developing solution containing nucleic acid (Example) and the case of using a developing solution not containing nucleic acid (Comparative Example), it is possible to measure AFP (alpha-fetoprotein) in a sample with higher sensitivity. did it. That is, it was revealed that a good S / N ratio can be obtained by using the developing solution of the present invention containing a nucleic acid molecule in an immunochromatographic method. In this experiment, all signals on the test line were confirmed.

Claims (7)

1. A developing solution in an immunochromatographic method using a detection reagent labeled with an insoluble carrier, comprising a nucleic acid molecule.
2. The developing solution according to claim 1, wherein the insoluble carrier is silica particles.
3. The developing solution according to claim 2, wherein the silica particles are those in which a fluorescent compound is chemically bonded or adsorbed.
4. The developing solution according to any one of claims 1 to 3, wherein the nucleic acid molecule is composed of one or more nucleotides.
5. An immunochromatographic method, wherein the developing solution according to any one of claims 1 to 4 is used as a developing solution constituting a mobile phase.
6. An immunochromatographic method using the developing solution according to any one of claims 1 to 4 as a sample diluent.
7. A detection kit for immunochromatography, comprising at least the developing solution according to any one of claims 1 to 4 and a chromatographic medium.